GeneChip Eukaryotic Double Strand Whole Transcript Protocol

Transcription

1 GeneChip Eukaryotic Double Strand Whole Transcript Protocol for Arabadopsis, C. elegans, Drosophila, and Yeast Tiling Arrays Protocol For Research Use Only. Not for use in diagnostic procedures.

2 Information in this document is subject to change without notice. DISCLAIMER TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT. Important Licensing Information These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses. Corporate entity Life Technologies Carlsbad, CA USA Toll free in USA Trademarks All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.all other trademarks are the property of their respective owners Thermo Fisher Scientific Inc. All rights reserved. P/N

3 Contents GeneChip TM Eukaryotic Double Strand Whole Transcript Protocol for Arabadopsis, C. elegans, Drosophila, and Yeast Tiling Arrays... 3 Safety Information... 3 Materials Required... 4 Reagents Required... 4 Instruments Required... 5 Eukaryotic Double Strand Whole Transcript Protocol... 6 Preparation of RNA-Random Primer Mix... 6 Second-Cycle, Second Strand cdna Synthesis and Cleanup... 8 Fragmentation, Labeling, and Hybridization GeneChip Eukaryotic Double Strand Whole Transcript Protocol

4 GeneChip TM Eukaryotic Double Strand Whole Transcript Protocol for Arabadopsis, C. elegans, Drosophila, and Yeast Tiling Arrays The GeneChip TM Eukaryotic Double Strand Whole Transcript Protocol is to be used in preparation of biotinlabeled ds cdna whole transcript target for hybridization to Arabidopsis, C. elegans, Drosophila and Yeast tiling arrays. This protocol replaces the former GeneChip TM Whole Transcript (WT) Double-Stranded Target Assay (No Amplification). For questions, please contact Technical Support. Safety Information Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. Caution: All chemicals should be considered as potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing, such as lab coat, safety glasses and gloves. Care should be taken to avoid contact with skin and eyes. In case of contact with skin or eyes, wash immediately with water. See SDS (Safety Data Sheet) for specific advice. GeneChip Eukaryotic Double Strand Whole Transcript Protocol 3

5 Materials Required Reagents Required Table 1 Reagents Required Material Source P/N crna Synthesis Part of the GeneChip TM WT PLUS Reagent Kit Consists of: WT Amplification Kit Module 1 WT Amplification Kit Module 2 GeneChip TM Poly-A RNA Control Kit cdna Synthesis Thermo Fisher GeneChip TM WT PLUS Reagent Kit ( or ) Random Primers, 3 μg/μl, 300 μg Life Technologies dntps, Set of Four (da, dc, dg, dt) 100 mm Solutions, 4 X 25 μmol Thermo Fisher PK dutp, 100 mm Solution, 25 μmol Thermo Fisher UM Nuclease-free Water Thermo Fisher RNase Inhibitor (Recombinant) Thermo Fisher Magnesium Chloride (MgCl2), 1M Thermo Fisher SuperScript TM II, 200 U/μL, 40,000 U (5X First Strand buffer and 0.1 M DTT included) Life Technologies Exonuclease-free Klenow, 10 U/μL Thermo Fisher RNase H, 5 U/μL Thermo Fisher Fragmentation and Labeling Uracil-DNA Glycosylase (UDG) (2 U/μL) USB Human Apurinic/Apyrimidinic Endonuclease 1 (APE 1) (Includes 10X APE 1 Reaction Buffer) Terminal Deoxynucleotidyl Transferase (rtdt), Recombinant, (30 U/μL) (5X TdT buffer included) USB USB DNA Labeling Reagent, DLR, 10 mm USB Optional: RNA 6000 Nano Kit* Agilent Technologies cdna Clean-up PrepEase DNA Clean-Up Kit Thermo Fisher Hybridization, Wash and Stain GeneChip TM Expression Hybridization Control Kit (Part of the GeneChip TM WT PLUS Reagent Kit) Thermo Fisher GeneChip TM WT PLUS Reagent Kit ( or ) GeneChip TM Hybridization, Wash, and Stain Kit Thermo Fisher * Or equivalent DNA and RNA sizing reagents. 4 GeneChip Eukaryotic Double Strand Whole Transcript Protocol

6 Instruments Required Table 2 Instruments Required Instrument Source P/N GeneChip TM Hybridization Oven 645 Thermo Fisher (110/220V) GeneChip TM Fluidics Station 450 Thermo Fisher GeneChip TM Scanner G Thermo Fisher (North America) (International) GeneChip TM AutoLoader with External Barcode Reader Microcentrifuge NanoDrop TM UV-Vis Spectrophotometer* Optional: 2100 Bioanalyzer Pipette Thermal Cycler Vortex Mixer 65 C heat block or oven for incubation of Nucleasefree Water during Purification Thermo Fisher Major Laboratory Supplier Thermo Scientific Agilent Technologies Major Laboratory Supplier Various Major Laboratory Supplier Major Laboratory Supplier (GCS G S/N 501) (GCS G S/N 502) * Or equivalent quantitation instrument. Or equivalent DNA and RNA sizing instrument. GeneChip Eukaryotic Double Strand Whole Transcript Protocol 5

7 Eukaryotic Double Strand Whole Transcript Protocol Start at Step 1 with either: 7 µg total RNA 7 µg purified crna obtained using the WT PLUS Reagent Kit (P/N or ). Preparation of RNA-Random Primer Mix 1. Mix crna with the Random Primers in a strip tube, as listed in Table 3 below. Table 3 crna-primer Mix Component total or crna (7 μg) Random Primers (3 μg/μl) Nuclease-free Water Volume in 1 Rxn variable 1.0 μl up to 8 μl Total Volume 8.0 µl 2. Flick-mix, and spin down the tubes. 3. Incubate for at 70 C for 5 minutes, at 25 C for 5 minutes, and then cool the samples at 4 C for at least 2 minutes. 4. Prepare dntp mix as described in Table 4. Table 4 dntp Mix Component datp, 100 mm dctp, 100 mm dgtp, 100 mm dttp, 100 mm dutp, 100 mm Nuclease-free Water Volume in 1 Rxn 5 μl 5 μl 5 μl 4 μl 1 μl 30 μl Total Volume 50 µl 5. In a separate tube, prepare a master mix as described in Table 5. 6 GeneChip Eukaryotic Double Strand Whole Transcript Protocol

8 Table 5 First Strand cdna Synthesis Component Volume in 1 Rxn Master Mix for 3.5 Rxns 5X 1st Strand Buffer 4.0 μl 14.0 μl DTT, 0.1 M 2.0 μl 7.0 μl dntp, 10 mm (4dTTP:1dUTP) 1.0 μl 3.5 μl (from Step 4 above) RNase Inhibitor (Recombinant) 1.0 μl 3.5 μl SuperScript II, 200 U/μL 4.0 μl 14.0 μl Total Volume 12.0 µl 42.0 µl 6. Transfer 12 µl of the First Strand Master Mix to the crna samples (the total reaction volume is 20 µl). Mix thoroughly by gently flicking the tubes a few times. Centrifuge briefly to collect the reactions at the bottom of the tube. 7. Incubate the reactions at 25 C for 5 minutes, at 42 C for 90 minutes, and at 4 C for at least 2 minutes. TIP: POTENTIAL STOPPING POINT. Store products at 20 C if desired. GeneChip Eukaryotic Double Strand Whole Transcript Protocol 7

9 Second-Cycle, Second Strand cdna Synthesis and Cleanup 1. In a separate tube, assemble a master mix as listed in Table 6. Table 6 Second Strand cdna Synthesis Component Volume in 1 Rxn Master Mix for 3.5 Rxns MgCl2*, 17.5 mm 8.0 μl 28 μl dntp, 10 mm (4 dttp:1dutp) 0.6 μl 2.1 μl (From Step 4 on page 6) Exonuclease-free Klenow, 10 U/μL 2.7 μl 9.5 μl Nuclease-free Water 8.5 μl 29.7 μl RNAse H, 5 U/ ul 0.2 μl 0.7 μl Total Volume 20 µl 70 µl * Make a fresh dilution of the MgCl 2 each time. Mix 2 µl of 1M MgCl 2 with 112 µl of Nuclease-free Water. If needed dilute RNase H in the appropriate storage buffer: 20mM Tris-HCl (ph 7.9), 100mM KCl, 10mM MgCl 2, 0.1mM EDTA, 0.1mM DTT, 50% glycerol. 2. Transfer 20 µl of the Second Strand Master Mix to the first strand reaction tubes (the total reaction volume is 40 µl). Mix thoroughly by gently flicking the tubes a few times. Centrifuge briefly to collect the reactions at the bottom of the tubes. 3. Incubate the reactions at 37 C for 40 minutes, at 75 C 10 minutes, and at 4 C for at least 2 minutes. TIP: POTENTIAL STOPPING POINT. Store products at 20 C if desired. 4. Proceed to ds cdna clean-up using the USB PrepEase DNA Clean-Up Kit: 1. Adjust DNA binding conditions. 1) Add 5 volumes of N2P Buffer to 1 volume of sample (e.g., 200 µl N2P Buffer and 40 µl sample). Mix well. 2. Bind DNA sample to column. 1) Place PrepEase TM Clean-Up Column into a 2 ml PrepEase TM Collecting Tube. 2) Pipet the sample directly into the center of the column. 3) Centrifuge 1 minute at 11,000 x g. 4) Discard flow-through. 3. Wash column. 1) Add 600 µl NT3 Buffer to column. 2) Centrifuge 1 minute at 11,000 x g. 3) Discard flow-through. Place column back into collecting tube. 4. Dry column. 1) Centrifuge 2 minutes at 11,000 x g. 5. Elute DNA. 1) Place the column into a clean 1.5 ml microcentrifuge tube. 8 GeneChip Eukaryotic Double Strand Whole Transcript Protocol

10 2) Add 50 µl NE Buffer to column. 3) Incubate at room temperature for 1 minute. 4) Centrifuge 1 minute at 11,000 x g. 5. Take 1 µl from each sample to determine the ds cdna yield by spectrophotometric UV measurement at 260 nm, 280 nm and 320 nm. Fragmentation, Labeling, and Hybridization 1. Fragmentation of ds cdna. 1. Fragment each of the samples as listed in Table 7 below: Table 7 Fragmentation of ds cdna Component Volume/Amount in 1 Rxn ds cdna Nuclease-free Water 10X APE 1 Reaction Buffer Uracil-DNA Glycosylase (2U/ μl) Human Apurinic/Apyrimidinic Endonuclease 1 (APE 1) 10 units/μl 7.5 μg up to 23.7 μl 4.8 μl 7.5 μl 12.0 μl Total Volume 48 µl 1) Flick-mix, and spin down the tubes. 2) Incubate the reactions at 37 C for 1 hour, at 93 C for 2 minutes, and at 4 C for at least 2 minutes. 3) Flick-mix, spin down the tubes, and remove 3 µl for fragmentation analysis using a Bioanalyzer. Please see the Reagent Kit Guide that comes with the RNA 6000 Nano LabChip Kit. The average peak size of the fragmented samples should be approximately 50 bp. 4) If not labeling the samples immediately, store the fragmented ds cdna at 20 C. 2. Labeling of fragmented ds cdna: 1. Prepare the labeling reactions for the samples as listed in Table 8 below. Table 8 Labeling of Fragmented ds cdna Component Fragmented ds cdna 5x TdT Reaction Buffer Terminal Deoxynucleotidyl Transferase (rtdt), Recombinant, (30 U/uL) DNA Labeling Reagent, DLR, 10 mm Volume in 1 Rxn 45.0 μl 12.0 μl 2.0 μl 1.0 μl Total Volume 60.0 µl 2. Flick-mix, and spin down the tubes. 3. Incubate the reactions at 37 C for 1 hour, at 70 C for 10 minutes, and at 4 C for at least 2 minutes. 4. Remove 4 µl of each sample for Gel-shift analysis (optional). GeneChip Eukaryotic Double Strand Whole Transcript Protocol 9

11 3. Hybridization of Fragmented and labeled ds cdna on the arrays (standard arrays). 1. Prepare the hybridization mix in a 1.5 ml RNase-free microfuge tube as shown in Table 9 below. Table 9 Hybridization Mix Component Volume in 1 Rxn Final Concentration or Amount Fragmented-labeled ds DNA 60.0 μl 37.5 ng/μl Control Oligonucleotide B2 3.3 μl 50 pm 20X Eukaryotic Hybridization 10 μl 1.5, 5, 25 and 100 pm, Controls (biob, bioc, biod, cre) respectively 2X Hybridization Mix 100 μl 1X DMSO 14 μl 7% Nuclease-free Water Up to 200 μl Total Volume µl 2. Flick-mix, and centrifuge the tube. 3. Heat the hybridization cocktail mix at 99 C for 5 minutes. Cool to 45 C for 5 minutes, and centrifuge at maximum speed for 1 minute. 4. Inject the appropriate amount of the specific sample (200 µl for 49-format arrays and 130 µl for 100-format arrays) into the array through one of the septa. 5. Place array in 45 C hybridization oven, at 60 rpm, and incubate for 16 (± 1.0) hours. 4. Wash/Stain arrays with Thermo Fisher TM Hybridization, Wash and Stain Kit using the FS450_0001 fluidics protocol for 49-format arrays and FS450_0002 fluidics protocol for 100-format arrays. 10 GeneChip Eukaryotic Double Strand Whole Transcript Protocol

12 Documentation and support Obtaining support Technical support For the latest services and support information for all locations, visit At the website, you can: Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions (FAQs) Submit a question directly to Technical Support (thermofisher.com/support) Search for user documents, SDSs, vector maps and sequences, application notes, formulations, handbooks, certificates of analysis, citations, and other product support documents Obtain information about customer training Download software updates and patches Safety Data Sheets (SDS) Limited product warranty Safety Data Sheets (SDSs) are available at thermofisher.com/support. Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at If you have any questions, please contact Life Technologies at GeneChip Eukaryotic Double Strand Whole Transcript Protocol 11

13 For support visit thermofisher.com/support or thermofisher.com 23 January 2017